In 1989, an endotoxin-induced serum activity that induced interferon-γ (IFN-γ) obtained from mouse spleen cells was described (Nakamura et al 1989). This serum activity did not function as a direct inducer of IFN-γ but rather as a co-stimulant together with IL-12, IFN-α/β, TNF or mitogens. An attempt to purify the activity from post-endotoxin mouse serum revealed an apparently homogeneous 50-55 kDa protein (Nakamura et al. 1993). Since other cytokines can act as co-stimulants for IFN-γ production, the failure of neutralizing antibodies to IL-1, IL-4, IL-5, IL-6, or TNF to neutralize the serum activity suggested it was a distinct factor. In 1995, the same scientists demonstrated that the endotoxin-induced co-stimulant for IFN-γ production was present in extracts of livers from mice preconditioned with P. acnes (Okamura at al. 1995). In this model, the hepatic macrophage population (Kupffer cells) expands and in these mice, a low dose of bacterial lipopolysaccharide (LPS), which in non-preconditioned mice is not lethal, becomes lethal. The factor, named IFN-γ-inducing factor (IGIF) and later designated interleukin-18 (IL-18), was purified to homogeneity from 1,200 grams of P. acnes-treated mouse livers. Degenerate oligonucleotides derived from amino acid sequences of purified IL-18 were used to clone a murine IL-18 cDNA (Okamura et al. 1995). Messenger RNAs for IL-18 and interleukin-12 (IL-12) are readily detected in activated macrophages. IL-18 does not induce IFN-γ by itself, but functions primarily as a co-stimulant with mitogens or IL-12. The human cDNA sequence for IL-18 was reported in 1996.
Interleukin IL-18 shares structural features with the IL-1 family of proteins (Nakamura et al. 1993, Okamura et. al 1995, Ushio et al. 1996 and Bazan et al. 1996). Unlike most other cytokines, which exhibit a four-helix bundle structure, IL-18 and IL-1β have an all β-pleated sheet structure (Tsutsui et al. 1996). Similarly to IL-1β, IL-18 is synthesized as a biologically inactive precursor (proIL-18), lacking a signal peptide (Ushio et al 1996). The IL-1β and IL-18 precursors are cleaved by caspase-1 (IL-1β-converting enzyme, or ICE), which cleaves the precursors after an aspartic acid residue in the P1 position. The resulting mature cytokines are readily released from the cell (Ghayur et al. 1997 and Gu et al. 1997).
IL-18 is a co-stimulant for cytokine production (IFN-γ, IL-2 and granulocyte-macrophage colony stimulating factor) by T helper type I (Th1) cells (Kohno et al. 1997) and also a co-stimulant for FAS ligand-mediated cytotoxicity of murine natural killer cell clones (Tsutsui et al. 1996).
Th1 lymphocytes are involved in the immune responses against tumors (Seki et al. 2000). Th1 responses include the secretion of the cytokines IL-2, IL-12, IL-18 and IFN-γ, as well as the generation of specific cytotoxic T lymphocytes recognizing specific tumor antigens. The Th1 response is also a vital arm of host defense against many microorganisms. However, the Th1 response can also be associated with non-desirable effects, such as the development of several autoimmune diseases, inflammation and organ transplant rejection.
Cytokine binding proteins (soluble cytokine receptors) are usually the extracellular ligand binding domains of their respective cell surface cytokine receptors. They are produced either by alternative splicing or by proteolytic cleavage of the cell surface receptor. These soluble receptors have been described in the past: for example, the soluble receptors of IL-6 and IFN-γ (Novick et al. 1989), TNF (Engelmann et al. 1989 and Engelmann et al. 1990), IL-1 and IL-4 (Maliszewski et al. 1990), IFN-α/β (Novick et al. 1994, Novick et al. 1992). One cytokine-binding protein, named osteoprotegerin (OPG, also known as osteoclast inhibitory factor—OCIF), a member of the TNFR/Fas family, appears to be the first example of a soluble receptor that exists only as a secreted protein (Anderson et al. 1997, Simonet et al. 1997, Yasuda et al. 1998).
An interleukin-18 binding protein (IL-18BP) was affinity purified, on an IL-18 column, from urine (Novick et al. 1999). IL-18BP abolishes IL-18 induction of IFN-γ and of IL-8, activation of NF-kB in vitro and induction of IFN-γ in vivo. IL-18BP is a soluble circulating protein, which is constitutively expressed in the spleen, and belongs to the immunoglobulin superfamily. The most abundant IL-18BP isoform, the spliced variant isoform a, exhibits a high affinity for IL-18 with a rapid on-rate and a slow off-rate, and a dissociation constant (Kd) of approximately 400 pM (Kim et al. 1999).
The residues involved in the interaction of IL-18 with IL-18BP have been described through the use of computer modelling (Kim et al. 1999) and based on the interaction of IL-1β with the IL1R type I (Vigers et al. 1997). In the model for IL-18 binding to the IL-18BP, the Glu (E) residue at position 42 and the Lys (K) residue at position 89 of IL-18 have been proposed to bind to Lys-130 and Glu-114 in IL-18BP, respectively (Kim et al. 1999).
IL-18BP is constitutively present in many cells (Puren et al. 1999) and circulates in healthy humans (Urushihara et al. 2000). The high affinity of IL-18BP to IL-18 as well as the high concentration of IL-18BP found in the circulation (20 fold molar excess over IL-18) represents a unique situation in cytokine biology. Therefore, it is presumed that most, if not all, of the IL-18 molecules in the circulation is bound to the IL-18BP. The circulating IL-18BP, which competes with cell surface receptors for IL-18, may act as a natural anti-inflammatory and an immunosuppressive molecule.
Viral agents encode IL-18BP like proteins, for example M. contagiosum viral proteins MC53 and MC54 share a significant homology to mammalian IL-18BP (Novick et al. 1999). M. contagiosum proteins MC53 and MC54 possess the ability to bind and neutralize human IL-18 in a fashion similar to that of IL-18BP (Xiang and Moss 1999). The ectromelia poxvirus p13 protein, which is homologous to IL-18BP, binds human IL-18 and inhibits its activity in vitro. Mice infected with a p13 deletion mutant virus exhibited decreased levels of infectivity (Born et al. 2000). Therefore, infectivity degree seems to correlate with the presence of viral IL-18BP.
The high levels of circulating IL18BP may represent a natural defense against excessive Th1 responses to infection and development of autoimmune diseases.
The cytokines of the IL-1-family, including IL-18, possess a variety of inflammatory and immunoregulatory properties during the first line and secondary response to infection (Dinarello 1996 and Nakanishi 2001). Six new members of the interleukin-1 (IL-1) gene family have been discovered from expressed sequence tag data base searches (Barton 2000, Busfield 2000, Debets 2001, Kumar 2000, Lin 2001, Mulero 1999, Pan 2001 and Smith 2000). These proteins share a common β-barrel pattern consisting of 12.beta.-strands and significant amino acid homology with the IL-1 receptor antagonist (IL-1Ra), IL-1β and IL-18. These new members of the IL-1 family are derived from a common ancestor as are IL-1 and IL-18 (Nicklin 2002, Taylor 2002). Except for IL-18, each maps to the same region on human chromosome 2 (Nicklin 2002, Mulero 2000, Taylor 2002 and Busfield 2000). Their biological function of these IL-1 homologues is presently unknown. Five different splice variants of the novel IL-1 homologue IL-1H4 (IL-IF7a-e) have been described (Busfield 2000, Kumar 2000, Pan 2001, Smith 2000, Taylor 2002). The first isoform described, IL-1F7a, has an unique N-terminus consisting of exon 3 of the IL-1 F7 gene, which is not present in the other splice variants of the gene. The short isoforms IL-IF7c, IL-IF7d and IL-IF7e lack exon 4,2 or both, respectively. Only IL-1F7b and c containing exon 1 and 2 express a N-terminal prodomain that has a potential caspase-1 (ICE) cleavage site(s) (Kumar 2002). In addition to these splice variants, amino acid polymorphisms (V31 G and A42T) exist in IL-1F7b based on two base pair mutations in exon 2 (Kumar 2000, Pan 2001). Despite extensive data base searches and sequencing of the IL-1-gene locus, no murine homologue of IL-1 H4 has yet been found. IL-1F7b shares significant sequence homology with IL-18. The hallmark for IL-18 activity is its ability to induce IFNγ in T-cells or natural killer (NK) cells in the presence of IL-2, IL-12 or IL-15 as costimulant. The activity of IL-18 is mediated via the IL-18R complex consisting of the ligand-binding chain termed IL-18R.alpha. (Torigoe 1997) and a signaling chain termed IL-18β (3 (Born 1998, Kim 2001). Upon binding to the IL-18Rα chain and formation of the hetero complex with the IL-18Rβ chain, IL-18 induces activation of IL-1 receptor-associated kinase and TNF receptor-associated factor 6 (TRAF-6). These activated kinases eventually result in the translocation of nuclear factor .kappa.-B (NF-κB) (Matsumoto, Robinson). IL-1F7b has been reported to bind to the IL-18R.alpha. using a receptor pulldown assay (Pan 2001) or computerchip-based binding studies (BIACORE®) (Mulero 2000). Significant but low affinity binding of Kd=130 mM was only observed for the mature form of IL-1F7b without the propeptide suggesting biological relevance to IL-1F7b processing by ICE (Kumar 2002). Despite the binding to the IL-18Rα, no IL-18-like or antagonistic activity of either pro or mature IL-1F7b was demonstrated (Pan 2001, Kumar 2002).
It has been suggested that interleukin IL-18 is involved in the progression of pathogenicity in chronic inflammatory diseases, including endotoxin shock, hepatitis, and autoimmune-diabetes (Kahiwamura and Okamura, 1998). A further indication of a possible role of IL-18 in the development of liver injury resulted from experiments published by Tsuij et al. (Tsuij et al., 1999), showing an elevated level of IL-18 in lipopolysaccharide-induced acute liver injury in a mouse model. However, the mechanism of the multi-functional factor IL-18 in the development of liver injury has not been elucidated so far.
Liver damage or injury may have diverse causes. It may be due to viral or bacterial infections, alcohol abuse, immunological disorders, or cancer, for example.
Viral hepatitis, due to Hepatitis B virus and Hepatitis C virus, for example, are poorly managed diseases that afflict large number of people world-wide. The number of known hepatitis viruses known is constantly increasing. Apart from Hepatitis B and C virus, at least four other viruses causing virus-associated hepatitis have been discovered so far, called Hepatitis A, D, E and G-Virus.
Alcoholic liver disease is another widespread disease associated with chronic consumption of alcohol. Immune hepatitis is a rare autoimmune disease that is poorly managed. Liver injury also includes damages of the bile ducts. Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by destruction of the intrahepatic bile ducts.
Several studies have demonstrated that damage to the liver in diseases such as alcoholic hepatitis, liver cirrhosis, viral hepatitis and primary biliary cirrhosis is associated with T-helper cell-1 (Th1) responses. In one study, a novel liver injury model was established in mice by targeting of ovalbumin-containing liposomes into the liver, followed by adoptive transfer of ovalbumin-specific Th1 cells. Combined treatment of mice with ovalbumin-containing liposomes and Th1 cell transfer caused an increase in serum transaminase activity that was paralleled with an elevation of serum IFN-γ levels. In sharp contrast, ovalbumin-specific Th2 cell transfer resulted in an increase of serum IL-4 levels but did not induce liver injury. The liver injury was blocked by anti-IFN-γ antibodies and anti-tumor necrosis factor (TNF)-α antibodies. These findings indicate that Th1 cells are the major effector cells in acute liver injury (Nishimura and Ohta, 1999) In another set of studies it was shown that mice over-expressing IFN-γ exhibit spontaneous hepatitis without any pathogen or any other stimulant (Okamoto et al., 1998).
Another study implicated Th1 responses in primary biliary cirrhosis (PBC). PBC is an autoimmune liver disease characterized by destruction of the intrahepatic bile ducts. It is generally believed that cellular immune mechanisms, particularly involving T cells, result in this bile duct damage. The relative strength of Th1 and Th2 responses has recently been proposed to be an important factor in the pathophysiology of various autoimmune diseases. In this study, the subset balance in PBC was evaluated by detection of cytokines specific to the two T-cell subsets, i.e., IFN-γ for Th1 cells and IL-4 for Th2 cells. IFN-γ and IL-4 messenger RNA (mRNA) positive cells were counted in liver sections from 18 patients with PBC and 35 disease controls including chronic active hepatitis C, extrahepatic biliary obstruction, and normal liver, using nonisotopic in situ hybridization and immunohistochemistry. Mononuclear cells expressing IFN-γ and IL-4 mRNA were aggregated in inflamed portal tracts in PBC livers, but were rarely present in extrahepatic biliary obstruction, alcoholic fibrosis, or normal liver sections. The IFN-γ and IL-4 mRNA positive cells in PBC livers were detected in significantly higher numbers than in control livers (P<0,01). Moreover, IFN-γ mRNA expression was more commonly detected than IL-4 expression in PBC livers, and the levels of IFN-γ mRNA expression were highly correlated with the degree of portal inflammatory activity. IFN-γ mRNA-positive cells were detected primarily around damaged bile ducts that were surrounded by lymphoid aggregates. The data indicate that Th1 cells are the more prominent T-cell subset in the lymphoid infiltrates in PBC (Harada et al., 1997).
The cytokine pattern on viral antigen recognition is also believed to exert a profound influence on the resolution of viral infections and viral clearance. One study investigated whether a cytokine imbalance oriented toward Th2 type response plays a role in chronic hepatitis B. Cytokine profiles of peripheral blood mononuclear cells associated with chronic hepatitis B were analyzed by RT-PCR. Upon hepatitis B surface antigen (HbsAg) stimulation, expression of IFN-γ, IL-2, IL-4, and IL-10 was detected in 41%, 8%, 41%, and 50% of the patients, respectively. Among these cytokines, the expression of the Th1 cytokine IFN-γ was associated with high levels of serum AST/ALT (Aspartate aminotransferase/Alanine aminotransferase), representing typical markers of liver damage. Th2 type cytokines were not shown to exert a protective effect on hepatocytes. In conclusion, production of a Th1 cytokine, IFN-γ, by HBsAg-reactive cells was associated with hepatocyte damage in chronic hepatitis B (Lee et al., 1999). High levels of the FAS ligand and its receptor (CD95) were reported in liver of hepatitis B patients (Luo et al., 1997). FAS ligand is considered to be one of the major cytotoxic agents leading to hepatocyte apoptosis.
Another study identified factors associated with the progression of liver injury in 30 hepatitis C virus/RNA (HCV/RNA)-positive untreated patients with chronic hepatitis. Necroinflammatory and architectural damage were evaluated using Ishak's score. Activated hepatic stellate cells (HSC) were visualized by immunohistochemistry for α-smooth muscle actin (αSMA) and quantitated by morphometry. Plasma HCV/RNA was evaluated using a competitive RT-PCR method. To study the type of immune response involved in the progression of liver injury, IFN-γ-positive cells (as expression of a Th1-like response) were evaluated by immunohistochemistry and quantitated by morphometry. It was found that HSC were mostly detected close to areas of lobular necroinflammation or lining fibrotic septa. The αSMA- and Sirius Red-positive parenchyma correlated significantly with necroinflammatory and architectural scores. IFNγ-positive cells were detected in periportal areas associated with the inflammatory infiltrates and significantly correlated with architectural damage. It was therefore concluded that HSC activation and progression of liver injury are associated with a Th1-like response (Baroni et al, 1999). Similarly to the case of Hepatitis B, FAS ligand and its receptor were found in liver and sera of hepatitis C patients (Hiramatsu et al, 1994; Okazaki et al, 1996; Lio et al., 1998)
Th1 cytokines and other Th1 markers were found to be associated with alcoholic hepatitis and liver cirrhosis. Inflammatory stimuli and lipid peroxidation activate nuclear factor κB (NF-κB) and upregulate proinflammatory cytokines and chemokines. In one study, the relationship between pathological liver injury, endotoxemia, lipid peroxidation, and NF-κB activation and imbalance between pro- and anti-inflammatory cytokines was evaluated. Rats (5 per group) were fed ethanol and a diet containing saturated fat, palm oil, corn oil, or fish oil by intragastric infusion. Dextrose isocalorically replaced ethanol in control rats. Pathological analysis was performed and measurements of endotoxin were taken, lipid peroxidation, NF-κB, and messenger RNA (mRNA) levels of proinflammatory cytokines (TNFα, IL-1beta, IFN-γ, and IL-12), C-C chemokines (regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein [MCP]-1, macrophage inflammatory protein [MIP]-1-α), C—X—C chemokines (cytokine induced neutrophil chemoattractant [CINC], MIP-2, IP-10, and epithelial neutrophil activating protein [ENA]-78), and anti-inflammatory cytokines (IL-10, IL-4, and IL-13). Activation of NF-κB and increased expression of proinflammatory cytokines C—C and C—X—C chemokines was seen in the rats exhibiting necroinflammatory injury (fish oil-ethanol and corn oil-ethanol). These groups also had the highest levels of endotoxin and lipid peroxidation. Levels of IL-10 and IL-4 mRNA were lower in the group exhibiting inflammatory liver injury. Thus, activation of NF-κB occurs in the presence of proinflammatory stimuli and results in increased expression of Th1 proinflammatory cytokines and chemokines (Naji et al., 1999). FAS ligand and its receptor are also elevated in alcoholic liver diseases, suggesting once again that Th1 cytokines are involved in the autoimmune processes induced in alcoholic hepatitis (Galle et al., 1995; Taieb et al, 1998; Fiore et al., 1999).
TNF-α has also emerged as a common pathway in the pathogenesis of alcohol-related hepatic necro-inflammation. Increased levels of hepatic and serum TNF have been documented in animal models of alcoholic liver disease and in human alcoholic liver disease. This dysregulated TNF metabolism has been postulated to play a role in many of the metabolic complications and the liver injury of alcoholic liver disease (Grove et al., 1997; McClain and Cohen, 1989). For instance it was found in one study that patients with alcoholic hepatitis had higher TNF-α levels (mean, 26.3 ng/L; 95% CI, 21.7 to 30.9) than normal subjects (6.4 ng/L; CI, 5.4 to 7.4). Patients who subsequently died had a higher TNF-α level (34.7 ng/L; CI, 27.8 to 41.6) than survivors (16.6 ng/L; CI, 14.0 to 19.2). In patients with alcoholic hepatitis, TNF-α levels correlated positively with serum bilirubin (r=0.74; P=0.0009) and serum creatinine (r=0.81; P=0.0003). Patients with alcoholic hepatitis had higher TNF-α levels than patients with inactive alcoholic cirrhosis (11.1 ng/L; CI, 8.9 to 13.3) and severely alcoholic persons without liver disease (6.4 ng/L; CI, 5.0 to 7.8). Patients with abnormal renal function had lower TNF-α levels (14.1 ng/L; CI, 5.4 to 22.8) than patients with alcoholic hepatitis. It was therefore concluded that elevations in TNF-α in alcoholic hepatitis are most marked in severe cases, suggesting that TNF-α plays a role in the pathogenesis (Bird et al., 1990).
TNF mediates many of the biologic actions of endotoxin. Recent studies have shown that TNF administration may cause liver injury and that TNF may mediate the lethality of the hepatotoxin galactosamine. One of the most potent TNF inducers is endotoxin. Because patients with alcoholic liver disease frequently have endotoxemia and because many of the clinical manifestations of alcoholic hepatitis are known biologic actions of TNF, its activity was evaluated in patients with alcoholic hepatitis. Basal and lipopolysaccharide-stimulated TNF release from peripheral blood monocytes, a major source of TNF production, was determined in 16 patients with alcoholic hepatitis and 16 healthy volunteers. Eight of 16 alcoholic hepatitis patients and only two of 16 healthy volunteers had detectable spontaneous TNF activity (p less than 0.05). After lipopolysaccharide stimulation, mean monocyte TNF release from alcoholic hepatitis patients was significantly increased to over twice that of healthy controls (25.3+/−3.7 vs. 10.9+/−2.4 units per ml, p less than 0.005). It was therefore concluded that monocytes from alcoholic hepatitis patients have significantly increased spontaneous and lipopolysaccharide-stimulated TNF release compared to monocytes from healthy volunteers (McClain and Cohen, 1989.
Lipopolysaccharide (LPS)-binding protein (LBP) and CD14 play key intermediary roles in the activation of cells by endotoxin. Gut-derived LPS has been postulated to participate in promoting pathological liver injury in alcoholic liver disease. It was demonstrated that rats fed intragastrically with ethanol in oil for 4 weeks had elevated levels of CD14 and LBP in their Kupffer cells and hepatocytes, respectively. Expression of CD14 mRNA was also elevated in nonmyeloid cells. Enhanced LBP and CD14 expression rapidly increases the LPS-induced expression of various pro-inflammatory cytokines and correlates with the presence of pathological liver injury in alcoholic liver injury (Su et al., 1998; Lukkari et al., 1999).
Arthritis is a disease involving joint inflammation. The joints show swelling, stiffness, tenderness, redness or warmth. The symptoms may be accompanied by weight loss, fever or weakness. When these symptoms last for more than two weeks, inflammatory arthritis e.g. rheumatoid arthritis may be the cause. Joint inflammation may also be caused by infection, which can lead to septic arthritis. A very common type of arthritis is degenerative joint disease (osteoarthritis).
The medicaments commonly prescribed for arthritis and related conditions are non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs include aspirin and aspirin-like drugs. They reduce inflammation, which is the cause for joint pain, stiffness and swelling of the joints. However, NSAIDs are unspecific drugs having a number of side effects, involving bleeding of the stomach (Homepage of the Department of Orthopaedics of the University of Washington on Arthritis, Frederick Matsen (Chairman), www.orthop.washington.edu). In addition to NSAIDs, Celebrex™, a cyclooxygenase (COX-2) inhibitor, is used to relieve the signs and symptoms of osteoarthritis and rheumatoid arthritis in adults. It is also indicated for the treatment of patients with familial adenomatous polyposis.
WO 01/00229 describes a combination of tumors necrosis factor (TNF) antagonists and COX-2 inhibitors for the treatment of inflammation.
TNF antagonists are also used for the treatment of arthritis. TNF antagonists are described, for example, in WO 9103553.
Studies indicate that the interleukin IL-18 plays a proinflammatory role in joint metabolism. Olee et al. (1999) showed that IL-18 is produced by articular chondrocytes and induces proinflammatory and catabolic responses. The IL-18 MRNA was induced by IL-1β in chondrocytes. Chondrocytes produced the IL-18 precursor and in response to IL-1 stimulation secreted the mature form of IL-18. Studies on IL-18 effects on chondrocytes further showed that it inhibits TGF-β-induced proliferation and enhances nitric oxide production. IL-18 stimulated the expression of several genes in normal human articular chondrocytes including inducible nitric oxide synthase, inducible cyclooxygenase, IL-6, and stromelysin. Gene expression was associated with the synthesis of the corresponding proteins. Treatment of normal human articular cartilage with IL-18 increased the release of glycosaminoglycans. These finding identified IL-18 as a cytokine that regulates chondrocyte responses and contributes to cartilage degradation.
The localisation of Interleukin-1β-converting enzyme (ICE)/caspase-1 in human osteoarthritic tissues and its role in the maturation of interleukin-1beta and interleukin-18 have been shown by Saha et al. (1999). Saha et al. studied the expression and production of caspase-1 in human normal and osteoarthritic (OA) cartilage and synovium, quantitated the level of ICE in OA chondrocytes, and examined the relationship between the topographic distribution of ICE, interleukin-1β (IL-1β), and IL-18, as well as apoptosis of chondrocytes. The experiments performed in this study indicated that ICE was expressed and synthesised in both human synovial membrane and cartilage, with a significantly greater number of cells staining positive in OA tissue than in normal tissue. ICE production was preferentially located in the superficial and upper intermediate layers of articular cartilage. The production of mature IL-1beta in OA cartilage explants and chondrocytes was completely blocked by treatment with a specific ICE inhibitor, which also markedly diminished the number of IL-18-positive cells. The relationship between active IL-1beta and ICE suggests that ICE may promote OA progression by activating this proinflammatory cytokine, and that IL-18 may play a role in cartilage pathology.
Gracie et al. (1999) suggested a proinflammatory role for IL-18 in rheumatoid arthritis. Gracie et al. detected the IL-18 mRNA and protein within rheumatoid arthritis synovial tissues in significantly higher levels than in osteoarthritis controls. It was also shown that a combination of IL-12 or IL-15 with IL-18 induced the IFN-γ production by synovial tissues in vitro. Furthermore, IL-18 administration of collagen/inclomplete Freund's adjuvant-immunized mice facilitated the development of an erosive, inflammatory arthritis, suggesting that IL-18 may be proinflammatory in vivo.
However, so far, apart from chemical compounds, only the blockade of TNFα and IL-1β by using soluble receptors or monoclonal antibodies have been shown to decrease murine collagen-induced arthritis (CIA, which is a mouse model for rheumatoid arthritis) (Williams et al., 1994), and were therefore suggested as a therapeutic for rheumatoid arthritis.
The term “chronic or idiopathic inflammatory bowel diseases” embraces at least two conditions: Crohn's disease and ulcerative colitis. Both are diseases of the gastrointestinal tract, Crohn's disease most commonly affecting the small bowel. When it also involves the colon, the differential diagnosis from ulcerative colitis (see below) can be a problem.
The chronic inflammation and ulceration in Crohn's disease usually starts with either small-intestinal obstruction or abdominal pain which may mimic acute appendicitis; other presentations can relate to its complications. The course of the disease is chronic, and there may be exacerbations and remissions in spite of therapy. Onset is usually in early adult life, with about half of all cases beginning between the ages of 20 and 30 years and 90% between 10 and 40 years. Slightly more males than females are affected.
Microscopy reflects the gross appearances. Inflammation involvement is discontinuous: it is focal or patchy. Collections of lymphocytes and plasma cells are found mainly in the mucosa and submucosa but usually affecting all layers (transmural inflammation). The classical microscopic feature of Crohn's disease is the presence of granule cells surrounded by a cuff of lymphocytes. The incidence of idiopathic inflammatory bowel diseases shows considerable geographic variation. These diseases have a much higher incidence in northern Europe and the United States than in countries of southern Europe, Africa, South America and Asia, although increasing urbanisation and prosperity is leading to a higher incidence in parts of southern Europe and Japan (General and Systematic Pathology, Churchill Livingstone, 3rd edition 2000, JCE Underwood, Ed.).
In Crohn's disease, clinically there are two main groups, the first comprising patients whose disease goes into lasting remission within three years of onset, the second comprising patients with disease persisting beyond three years.
Whatever the aetiology, there is evidence of persistence and inappropriate T-cell and macrophage activation in Crohn's disease with increased production of pro-inflammatory cytokines, in particular interleukins (IL) 1, 2, 6 and 8, Interferon (IFN)-γ and Tumor Necrosis Factor (TNF) α. Crohn's disease is characterised by sustained (chronic) inflammation accompanied by fibrosis. The process of fibroblastic proliferation and collagen deposition may be mediated by transforming growth factor β, which has certain anti-inflammatory actions, namely fibroblast recruitment, matrix synthesis and down-regulation of inflammatory cells, but it is likely that many other mediators will be implicated.
Ulcerative colitis is a non-specific inflammatory disorder of the large intestine, usually beginning in the rectum and extending proximately to a varying extent. Unlike Crohn's disease, ulcerative colitis is confined to the large intestine.
There is growing evidence to indicate that ulcerative colitis is a consequence of altered autoimmune reactivity but mucosal injury could also result from inappropriate T-cell activation and indirect damage brought about by cytokines, proteases and reactive oxygen metabolites from macrophages and neutrophils. This latter mechanism of damage to the colonic epithelium has been termed “innocent bystander” injury. Evidence in favour of autoimmunity is the presence of self-reactive T-lymphocytes and auto-antibodies directed against colonic epithelial cells and endothelial cells, and anti-neutrophil cytoplasmic auto-antibodies (ANCA). However, ulcerative colitis should not be thought of as an autoimmune disease in which mucosal injury is a direct consequence of an immunological reaction to self-antigens (General and Systematic Pathology, supra).
With regard to the therapy of Crohn's disease, most people are first treated with drugs containing mesalamine, a substance that helps control inflammation. Patients who do not benefit from it or who cannot tolerate it may be put on other mesalamine-containing drugs, generally known as 5-ASA agents. Possible side effects of mesalamine preparations include nausea, vomiting, heartburn, diarrhea, and headache.
Some patients take corticosteroids to control inflammation. These drugs are the most effective for active Crohn's disease, but they can cause serious side effects, including greater susceptibility to infection.
Drugs that suppress the immune system are also used to treat Crohn's disease. Most commonly prescribed are 6-mercaptopurine and a related drug, azathioprine. Immunosuppressive agents work by blocking the immune reaction that contributes to inflammation. These drugs may cause side effects like nausea, vomiting, and diarrhea and may lower a person's resistance to infection. When patients are treated with a combination of corticosteroids and immunosuppressive drugs, the dose of corticosteriods can eventually be lowered. Some studies suggest that immunosuppressive drugs may enhance the effectiveness of corticosteroids.
The U.S. Food and Drug Administration has approved the drug infliximab for the treatment of moderate to severe Crohn's disease that does not respond to standard therapies (mesalamine substances, corticosteroids, immunosuppressive agents) and for the treatment of open, draining fistulas. Infliximab, the first treatment approved specifically for Crohn's disease, is an anti-tumor necrosis factor (TNF) monoclonal antibody. Anti-TNF removes TNF from the bloodstream before it reaches the intestines, thereby preventing inflammation.
Antibiotics are used to treat bacterial overgrowth in the small intestine caused by stricture, fistulas, or prior surgery. For this common problem, the doctor may prescribe one or more of the following antibiotics: ampicillin, sulfonamide, cephalosporin, tetracycline, or metronidazole.
Diarrhea and crampy abdominal pain are often relieved when the inflammation subsides, but additional medication may also be necessary. Several anti-diarrheal agents could be used, including diphenoxylate, loperamide, and codeine. Patients who are dehydrated because of diarrhea are usually treated with fluids and electrolytes.
There remains to be a need for effective therapy for the treatment and/or prevention of inflammatory bowel diseases, in particular Crohn's disease (CD) and ulcerative colitis (UC), which have reduced side effects or are ideally even free of side effects.
Both histological and immunological observations indicate that cell-mediated immunity and T cell activation are key features of CD. Studies from humans and experimental models suggest that, in CD, the local immune response tends to be predominantly Th1 in type (Desreumaux et al, 1997) and that locally released cytokines, such as IFN-γ, IL-1β, and TNF-α, contribute to promote and expand the inflammatory response (Reimund et al, 1996).
The cytokine IL-18 plays an important role in Th1 mediated immune response in collaboration with the cytokine IL-12 by stimulating IFN-γ secretion, enhancing natural killer cell cytotoxicity, and stimulating TH1 cell differentiation (Uschito et al, 1996).
IL-18 acts together with IL-12, IL-2, antigens, mitogens, and possibly further factors, to induce the production of IFN-γ. IL-18 also enhances the production of GM-CSF and IL-2, potentiates anti-CD3 induced T cell proliferation, and increases Fas-mediated killing of natural killer cells. Mature IL-18 is produced from its precursor by the IL-1β converting enzyme (ICE, caspase-1). The IL-18 receptor consists of at least two components, co-operating in ligand binding. High- and low-affinity binding sites for IL-18 were found in murine IL-12 stimulated T cells (Okamoto et al., 1998), suggesting a multiple chain receptor complex. Two receptor subunits have been identified so far, both belonging to the IL-1 receptor family (Okamoto et al., 1999). The signal transduction of IL-18 involves activation of NF-κB (Matsumoto et al, 1997).
Recently, IL-18 has been suggested to have some implication in Inflammatory Bowel Diseases (Pizarro et al, 1999; Monteleone et al, 1999).
Pizarro et al. (1999) characterised the expression and localisation of IL-18 in colonic specimens and isolated mucosal cell populations from patients with Crohn's disease. Using a semiquantitative RT-PCR protocol, IL-18 mRNA transcripts were found to be increased in freshly isolated intestinal epithelial cells and lamina propria mononuclear cells from CD compared with ulcerative colitis and noninflamed control patients. IL-18 mRNA transcripts were more abundant in intestinal epithelial cells compared with lamina propria mononuclear cells. Immunohistochemical analysis of surgically resected colonic tissues localised IL-18 to both lamina propria mononuclear cells (specifically, macrophages and dendritic cells) as well as intestinal epithelial cells. Western blot analysis revealed that an 18,3-kDa band, consistent with both recombinant and mature human IL-18 protein, was found predominantly in CD vs UC intestinal mucosal biopsies; a second band of 24 kDa, consistent with the inactive IL-18 precursor, was detected in non inflamed areas from both CD and UC biopsies and was the sole form found in noninflamed controls.
Monteleone et al. (1999) confirmed these findings. Whole mucosal intestinal tissue and lamina propria mononuclear cells of 12 Crohn's disease and 9 ulcerative colitis patients and 15 non-inflammatory bowel disease controls were tested for IL-18 by semiquantitative RT-PCR and Western blot analysis. Transcripts for IL-18 were found in all samples tested. However, increased IL-18 mRNA accumulation was detected in both mucosal and lamina propria mononuclear cells samples from Crohn's disease in comparison to ulcerative colitis and controls. In Crohn's disease, transcripts for IL-18 were more abundant in the mucosal samples taken from involved areas. An 18-kDa band consistent with mature IL-18 was predominantly found in Crohn's disease mucosal samples. In mucosal samples from non-IBD controls, IL-18 was present as the 24-kDa polypeptide. Consistently, active IL-1beta-converting enzyme (ICE) subunit (p20) was expressed in samples from either CD or UC, whereas, in colonic mucosa from non-IBD controls, ICE was synthesised as precursor (p45) only.
Ohta et al. 2001 showed that the expression of IL-18 was increased in psoriatic lesional skin relative to that in normal skin. Their findings indicate that keratinocyte-derived IL-18 participates in the development of the Th1 response in psoriatic lesions, and that its bioactivity appears to be tightly regulated in cutaneous inflammation.
In several animal models, antibodies that neutralize endogenous IL-18 reduce the severity of disease. Endotoxin lethality is prevented by anti-IL-18. Even in models that are interferon-γ independent, neutralization of IL-18 prolongs survival. Anti-IL-18 also protects the liver against cellular injury induced by toxins or activated T cells. In models of hepatic melanoma metastasis, IL-18 blockade reduces the adherence of malignant cells by preventing IL-18 upregulation of vascular endothelial adhesion-1 molecule expression. IL-18 and IL-12 act synergistically to stimulate I cells and natural killer cells to produce IFN-gamma but neutralization of IL-18 prevents IL-12 induction of IFN-gamma. IL-18, like several cytokines, can be used to enhance host defense against tumors in mice a mechanism that is most of ten IFN-gamma-dependent. Nevertheless, it is the proinflammatory portfolio of IL-18, which likely contributes to enhance host defenses. In models or arthritis, lung injury or inflammatory bowel disease, neutralization of IL-18 reveals the important role of this cytokine in mediating inflammation (Dinarello 2000).
Published data imply that IL-18 may play a phatological role in inflammatory CNS diseases. Neutralization of IL-18 was shown to protect from brain injury (Yatsiv et al. 2002), ischemic injury (Mallat et al. 2002), cardiac dysfunction (Raeburn 2002) and neuritis (Yu et al 2002) in animal models.
However, there is evidence that IL-18 promotes host defense against tumors in mice. For example, in syngeneic mice, murine mammary carcinoma cells expressing murine IL-12 or murine IL-18 were less tumorogenic and formed tumors more slowly than did control non-expressing cells (Coughlin et al. 1998). Antibody neutralization studies revealed that the antitumor effects required IFN-γ. In a study by Tasaki it has been observed protective immunity induced in murine colon carcinoma cells by the expression of interleukin-18. Colon cancer cells transduced with vectors encoding the IL-18 gene could not form subcutaneous tumors when introduced in immunocompetent mice, and became resistant to non-transduced inoculated colon cancer cells. Immunohistochemical analysis revealed that the numbers of blood vessels in colon tumors with cells transduced with IL-18 vectors were markedly reduced. The loss of tumorigenicity of colon IL-18 transduced cells was not observed in immunocompromised mice. Thus, the IL-18 secreted from tumor cells acts as an adjuvant since it stimulates T helper type 1 cells to induce antitumor response (Tasaki et al 2000).
It has been suggested that IFN-α, exerts its anti-inflammatory action in vivo in chronic hepatitis C patients inter alia by induction of IL-18BP (Kaser et al. 2002).
In previous work it was found that compared with healthy individuals, the levels of the IL-18BP are markedly elevated in many diseases such as in sepsis (Novick 2001) in Acute Graft versus Host Disease (Zecchina 2001), in Crohn's disease (Corbaz 2002). However it has been found also that in these patients the levels of IL-18 in the circulation are very high and therefore the levels of IL-18BP present in the circulation may not be sufficient for complete neutralization of IL-18.
Thus, is therefore a need to provide means to treat and/or prevent diseases in which a cytokine from the IL-1 family such as IL-18 is involved in their pathology.